The chemokine receptor CXCR4 is an essential GPCR that has been implicated in a number of human diseases including HIV, WHIM syndrome, and cancer. The detailed mechanisms involved in the regulation of CXCR4 function and its role in cancer are currently poorly understood. Agonist- dependent signaling of GPCRs is principally regulated by GPCR kinases (GRKs) and arrestins with GRK phosphorylation of an activated GPCR often being the initial step in the regulatory process. CXCR4 is rapidly phosphorylated following agonist activation, although the specific sites of phosphorylation, the kinases involved, and the functional roles are not well defined. In this application, we propose to use molecular, biochemical, and cellular strategies to better characterize the mechanisms that regulate CXCR4 expression and function in normal and cancer cells. Our initial studies suggest that GRKS may play the major role in agonist-specific phosphorylation of CXCR4 in HEK293 cells. In addition, enhanced Ca2+ mobilization is observed when GRKS, GRK6, arrestin-2, or arrestin-3 are knocked down while ERK1/2 activation is enhanced by the loss of GRK2 but decreased by the loss of GRKS, GRK6, arrestin-2, or arrestin-3. These initial studies suggest that GRKs and arrestins may differentially regulate CXCR4 signaling. We will test the hypothesis that site-specific phosphorylation of CXCR4 mediates the kinetics and specificity of signaling by addressing several important questions. What specific residues in CXCR4 are phosphorylated in response to agonist stimulation and what kinases mediate site-specific phosphorylation? What are the functional roles of CXCR4 phosphorylation and what mechanisms underlie the functional effects of site-specific phosphorylation? Are any of these mechanisms dysfunctional in cancer? Our studies should provide important insight into the mechanisms involved in CXCR4 regulation as well as provide potential therapeutic approaches for controlling CXCR4 function.